The present invention relates to metallo-endopeptidases, sometimes also called neutral proteases, that are produced, processed and secreted by e.g. members of the bacterial genus Bacillus. The present invention provides genes encoding variants of metallo-enopeptidases that have been engineered to be resistant to prolonged boiling while maintaining their enzymatic performance at much lower temperatures. In addition, thermal stability of the metallo-endopeptidases is highly dependent on calcium at concentrations in the mM range. The invention thus further provides active metallo-endopeptidase variants whose stability depending on calcium concentration can be changed so as to provide metallo-endopeptidases that are calcium dependent or independent. The invention also provides genes that encode boiling-resistant metallo-endopeptidases whose stability depending on calcium concentration can be changed. The invention also provides vectors and cells comprising these genes and proteases produced through these genes, vectors and/or cells.
Denaturation of proteins at elevated temperatures is usually the result of unfolding which is followed by an irreversible process, most often aggregation. The notion that the unfolding processes involved in irreversible denaturation often have a partial (as opposed to global) character has been confirmed experimentally in several cases. We have studied the thermal stability and denaturation of a broad-specificity metalloprotease produced by Bacillus stearothermophilus CU21 (called TLP-ste) which shares 85% sequence identity with its more stable and better known counterpart Bacillus thermoproteolyticus (hereinafter also referred to as xe2x80x9cthermolysinxe2x80x9d). Thermolysin-like proteases (TLPs) are a family of homologous metalloproteases or neutral proteases that contain a catalytically important zinc ion in their active site. Thermal denaturation of thermolysin-like proteases (TLPs) depends on partial unfolding processes as well, which, however, are not followed by aggregation but by autolytic degradation starting at unknown sites in the partially unfolded molecule (Vriend, G. and Eijsink, V. G. H. J. Comput.-Aided Mol. Des. 7, 367-396, 1993). An extensive mutation study in which residues in TLP-ste were replaced via site-directed mutagenesis by the corresponding amino acid in thermolysin showed that only a few of the 43 substitutions between the two enzymes are important for stability (Veltman, O. R., Vriend, G., Middelhoven, H., Van den Burg, B., Venema, G. and Eijsink, V. G. H. Protein Engn. 9, 1181-1189, 1996). All important substitutions are clustered in the N-terminal domain of the protein, in particular in a weak region comprising the 55-69 surface loop (European patent application 94200182.7). Remarkably, stabilizing substitutions or combinations of several substitutions either (i) at the positions of SEQUENCE ID No. 2: 4, 56, 58, 63, 65 or 69 (for example A4T, T56A, G58A, T63F, S65P, A69P) or (ii) the positions of SEQUENCE ID No. 1: 4, 59, 61, 66, 68 or 72 (for example A4T, T59A, G61A, T66F, S68P, A72P) result in enzyme variants that are more stable than thermolysin. The three-dimensional structure of thermolysin is known (Holmes, M. A. and Matthews, B. W. (1982) J. Mol. Biol, 160, 623-639) and this enzyme was shown to bind four calcium atoms which contribute to thermal stability. Two calcium ions are bound in the so-called double-calcium binding site (Ca1,2), that is composed of ligands that are conserved in all TLPs. The other, single binding sites (Ca3 and Ca4) are composed of ligands that are conserved only in the more stable TLPs such as thermolysin and the TLP produced by B. stearothermophilus (TLP-ste). At elevated temperatures, TLPs are irreversibly inactivated as a consequence of autolysis. Autolysis follows first-order kinetics because its rate is determined by local unfolding processes that render the protease susceptible to autoproteolytic cleavage (Eijsink, V. G. H., Van den Burg. B., Vriend, G., Berendsen, H. J. C. and Venema, G. (1991) Biochem. Internatl. 24, 517-525). In their studies on the contribution of calcium ions to thermolysin stability, Dahlquist et al. (Dahlquist, F. W., Long, J. W, and Bigbee, W. L. (1976) Biochemistry 15, 1103-1111. ) and Roche and Voordouw (Roche, R. S. and Voordouw, G. (1978) CRC Crit. Rev. Biochem. 5, 1-23) concluded that the initial steps in thermal inactivation are accompanied by the release of one calcium ion (Ca3 or Ca4). Extensive mutagenesis studies of the TLP-ste have shown that a region near the Ca3 site is crucial for thermal stability (Veltman, O. R., Vriend, G., Middelhoven, P. J., Van den Burg, B., Venema, G. and Eijsink, V. G. H. (1996) Protein Engng. 9, 1181-1189. ). Thus, thermal inactivation seems to be dominated by one single xe2x80x98weakxe2x80x99 region, near Ca3. Considering the expected high structural similarity between thermolysin and TLP-ste (85 percent sequence identity) the studies on TLP-ste suggest that the critical calcium ion is Ca3 rather than Ca4.
Metallo endopeptidases can be applied in several industrial processes, for instance in the preparation of the artificial sweetener aspartame, but also in the leather industry, for dehairing or dewooling, in breweries and in the production of (poly)peptide or protein hydrolysates. These processes may need to be performed at high temperatures to accelarate the processes. However, the enzymes used normally are not resistant to elevated temperatures. Proteases that are more stable at higher temperatures can for example be obtained via mutations in the 55-69 area, however, they still do not withstand boiling for periods lasting longer than several minutes. On the other hand, boiling is a simple method to denature the enzyme and thus a method to stop the enzymatic process. Thus, there is a need to develop enzymes that are more resistant to the temperatures found at boiling for prolonged periods in a watery environment, with an half-life  greater than 0.5 hour at 100xc2x0 C. but that on the other hand, if needed, may be controlled by other methods to stop the enzymatic processes.
The invention provides strategies to design and construct genetically engineered thermolysine-like proteases that are far more resistant to boiling than those known before. The invention also provides the proteases resulting from such strategies as well as the use of said products in industrial processes and intermediates in making the products. The invention provides mutations leading to the introduction of disulfide bridges stabilizing the area in metallo-endopeptidases that is most susceptible to unfolding after heating which area is hereinafter referred to as the xe2x80x9cfunctional part of the sequencexe2x80x9d. In a first embodiment, the invention provides a recombinant DNA molecule comprising a least a functional part of the sequence of FIG. 7 and coding functional part of the sequence of FIG. 7A [SEQUENCE ID No. 1] or FIG. 7B [SEQUENCE ID No. 2] or FIG. 7 (which combines the SEQUENCE ID No. 1 and the SEQUENCE ID No. 2 as more fully explained, infra) coding wherein at least one codon is mutated to code for cysteine to generate a stabilizing disulfide bridge, and a polypeptide derived of said DNA molecule. For example, an important mutation concerns the introduction of a disulfide bridge cross-linking residue 60 [in SEQUENCE ID No. 2] or residue 63 [in SEQUENCE ID No. 1] in the critical region with residue 8 (common to each of SEQUENCE ID No. 1 and SEQUENCE ID No. 2) in the underlying xcex2-hairpin. For example, the invention provides a mutated TLP-ste variant with a half life at 100xc2x0 C. of almost 3 hours which is more than 1000 times that of the wild-type while it maintained its specific activity at 37xc2x0 C. Furthermore, the invention provides mutants that are active and stable in the presence of high concentrations of denaturing agents and which cleavage specificity at both moderate and high temperatures is largely unaffected by the stabilizing mutations.
The invention also provides calcium-dependent andxe2x80x94independent variants of thermolysine-like proteases. Another embodiment of the invention is a recombinant DNA molecule comprising a least a functional part of the sequence of FIG. 7B (SEQUENCE ID No. 2) or FIG. 7A (SEQUENCE ID No. 1) coding for a polypeptide having metallo-endopeptidase activity wherein at least one codon is mutated to code for an amino-acid providing the resulting gene product (polypeptide) with a reduced capacity to bind with calcium.
For example, (i) referring to FIG. 7B (SEQUENCE ID No. 2), the Ca3 binding site of TLP-ste was deteriorated by mutating one of the main ligands (Asp57), but substitutions T63F or A69P leave the calcium-dependent stability intact, and (ii) referring to FIG. 7A (SEQUENCE ID No. 1), the Ca3 binding site of TLP-ste was deteriorated by mutating one of the main ligands (Asp60) but substitutions T66F or A72P leave the calcium-dependent stability intact. Subsequently, the loss in stability is compensated for by introducing stabilising mutations in the direct environment of the Ca3 site. The results confirm the importance of the Ca3 site for stability and they show the feasibility of engineering various grades of calcium-dependency in otherwise stable variants.
In addition, by combining above substitutions, the invention provides variants that encode boiling-resistant metallo-endopetidases whose stability depending on calcium concentration can be changed. For example, the invention further provides a recombinant DNA molecule wherein (i) in SEQUENCE ID No. 2 at least one codon coding for A at position 4, or T at 56, or G at 58, or T at 63, or S at 65, or A at 69 is mutated; for example, wherein at least one codon encoding the amino acid at position 4 is replaced by a codon encoding T, or at position 56 by a codon encoding A, or at position 58 by a codon encoding A, or at position 63 by a codon encoding F, or at position 65 by a codon encoding P, or at position 69 by a codon encoding P, and (ii) in SEQUENCE ID No. 1 at least one codon coding for A at position 4, or T at 59, or G at 61, or T at 66, or S at 68, or A at 72 is mutated; for example, wherein at least one codon encoding the amino acid at position 4 is replaced by a codon encoding T, or at position 59 by a codon encoding A, or at position 61 by a codon encoding A, or at position 66 by a codon encoding F, or at position 68 by a codon encoding P, or at position 72 by a codon encoding P. In yet another embodiment of the invention, a recombinant DNA molecule is provided wherein, referring to each of SEQUENCE ID No. 1 and SEQUENCE ID No. 2, the codon coding for G at position 8 has been modified to code for cysteine (C) or wherein (i) referring to SEQUENCE ID No. 2 the codon coding for D at position 57 has been mutated to code for serine, or (ii) referring to SEQUENCE ID No. 1 the codon coding for D at position 60 has been mutated to code for serine. The invention also provides a vector for expression of a polypeptide having metallo-endopeptidase activity, and provides a host cell expressing said polypeptide, comprising said vector. A polypeptide expressed by such a host cell has a high metallo-endopeptidase activity, especially when compared with conventionally used enzyme preparations, as for example shown in the experimental part of this description. One polypeptide as provided by the invention has for example a metallo-endopeptidase activity having a half-life in a watery environment at 100xc2x0 C. that is  greater than 0.5 hours. It goes without saying that such a high-active and stable enzyme has a wide range of uses, for example for the production of (poly)peptides or protein hydrolysates.
Industrial proteins, such as derived from soy, rice, milk, yeast, gluten, and other dietary proteins (such as animal food, fish protein) can now more easily be hydrolised or predigested, for example to increase its dietary value. Also, an hydrolysing enzyme as provided by the invention can be used to help clean (by hydrolysis) medical instruments under relatively mild conditions. Yet another use as provided by the invention is in cleaning (industrial) membranes and filters, in cleaning heat exchangers or condensers, or in any other instrument where deposits of proteins can be found that need removal. Specific hydrolysis, as provided by an enzyme provided by the invention is a new effective way of removing protein for example during waste water treatment. Small scale use is possible in cleaning contact lenses, surgical implants, and the like. In the laboratory, glassware and intruments can be cleaned more easily. Also preparation of DNA, freeing it from contamination protein in samples wherein nucleic acid is sought, is now more readily feasible. An enzyme provided by the invention can be used in pure or isolated form, or mixed with other components, such as with a detergent, or in washing powders, to provide synergistic action. Use as provided by the invention can for example be in the production of aspartame, for the production of leather, for dehairing or dewooling or in detergents or washing powders. Yet other examples are use for treatment of slaughter offal, optionally for the preparation of protein hydrolysates for commercial purposes. The invention provides use of a polypeptide as provided by the invention in hydrolysing protease resistant proteins. An example of such a use of a polypeptide provided by the invention is in hydrolysis of proteins that are more than an average protein resistant to hydrolysis. One example is hydrolysis of protease resistant xcex1-amylase by a polypeptide as provided by the invention, another example is hydrolysis of prion protein, which is also very resistant to proteolytic cleavage. Prion protein hydrolysis for example is very beneficial in cleaning medical instruments or testing apperature, without having to resort to extreme chemical or temperature treatments. Also, pretreatment of clinical samples in which prion protein may be detected (i.e. by ELISA) can now also be done more easily, whereby varying the assay conditions allows for early digestion of contaminating proteins, leaving the prion protein relatively intact. Because of its high activity and stability at higher temperatures, such a use of a polypeptide is preferred. This allows for rapid processes or processing of a large amount per treatment. For instance during pasteurisation or sterilisation treatment of food, contaminants in food, such as micro-organisms, or the above mentioned prion protein, or other proteinaceous contaminants can be rendered uninfectious by treating said food or feed (for both human or animal consumption) (possibly containing any of above mentioned proteinaceous contaminants) with a polypeptide provided by the invention. It is even possible to shorten temperature treatment because of the high activity of said polypeptide.
2. Materials and Methods
2.1 Production and Characterisation of Mutated Enzymes
Cloning, sequencing, sub-cloning, and expression of the TLP-ste gene (from strain B. stearothermophilus CU21), as well as production, purification and subsequent characterization of wild-type and mutant TLP-ste were performed as described earlier (Eijsink, V. G. H., Vriend, G., Van der Vinne, B., Hazes, B., Van den Burg, B. and Venema, G. (1992) Proteins 14, 224-236). Thermal stability was measured, using varying CaCl2concentrations in the standard assay buffer (20 mM Na acetate, pH 5.3, 0.01% Triton X-100, 0.5% isopropanol, 62.5 mM NaCl). T50 is the temperature of incubation at which 50 percent of the initial proteolytic activity is lost during a 30 minutes incubation.
The kinetic parameter kcat/Km (at 37xc2x0 C.) for the substrate 3-(-2-furylacryloyl)-L-glycyl-L-leucine-amide (FaGLa, Sigma Chemical Company, St. Louis, Mich., USA) was determined according to the method of Feder et al. (Feder, J. (1969) Biochemistry 6, 2088-2093), in a buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 5% or 1% DMSO, 1% isopropanol and 125 mM NaCl, using an 100 mM substrate concentration. Activities were derived from the decrease in absorption at 345 nm, using a Deof xe2x88x92317 Mxe2x88x921 cmxe2x88x923.
Specific activities for TLP-ste and the 8-fold mutant were determined using casein (0.8%) as a substrate in 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2 at 37xc2x0 C. The kcat/Km values for the enzymes were determined for two different furylacryloylated dipeptides as substrates, at 37xc2x0 C. in a thermostatted Perkin Elmer Lambda 11 spectrophotometer, in a total volume of 1 ml of 50 mM MOPS [4-morpholine-ethanesulfonic acid] (pH 7.0), 5 mM CaCl2, 5% or 1% DMSO, 0.5% isopropanol, 0.01% Triton X-100, 50 mM NaCl, with 100 mM of substrate, by measuring the decrease in absorption at 345 nm (Î345=xe2x88x92317 Mxe2x88x921xc2x7cmxe2x88x921). Stock solutions of the furylacryloylated dipeptides, (Sigma, St. Louis, Mich.) were prepared by dissolving 3-(-2-furylacryloyl)-L-glycyl-L-leucine amide (FaGLa) and 3-(-2-furylacryloyl)-L-alanyl-L-phenylalanine amide (FaAFa) in DMSO. The kcat/Km ratios of the enzymes were determined by varying the enzyme concentrations (over a 50 fold range) under pseudo-first order conditions by measuring the initial activity, essentially according to the method described by Feder30. The Ki for phosphoramidon (N-[a-L-rhamnopyrano-syl-oxyhydroxyphophinyl]-L-leucyl-L-tryptophan) was determined by a 30 minute preincubation of a 100 pM protease solution with varying concentrations of the inhibitor (10xe2x88x928 to 10xe2x88x923 M), in 50 mM MOPS (pH 7.0), 5 mM CaCl2, 50 mM NaCl, prior to the addition of the furylacryloated substrate. For determination of the Ki FaAFa was used as the substrate. Since the Km of the substrate for the enzyme is higher than the concentration used, IC50 values were taken to be equal to Ki values. The optimum temperature for activity was determined by incubating proteases with casein (0.8%) in 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, at different temperatures for 30 minutes, after which the amount of released peptides, indicative for activity, was measured11.
Protease activities were determined in 50 mM MOPS (pH 7.0), 5 mM CaCl2, 50 mM NaCl, 0.01% Triton X-100, 0.5% isopropanol, using 100 xcexcM FaGLa (3-(-2-furylacryloyl)-L-glycyl-L-leucine amide) as a substrate at 50xc2x0 C. Enzymes and denaturing agents were preincubated in the reaction mixture at 50xc2x0 C. for 15 minutes prior to the addition of the substrate. The reaction was followed by measuring the change in absorbance at 345 nm. The change in absorbance was linear during the time of measurement (30-60 minutes), indicating that the proteases were stable in this time interval and that substrate depletion was negligible. Activities are expressed as percentage of the activity in the absence of denaturant.
2.2 Structure Analysis
TLP-ste and thermolysin have 85% sequence identity which allowed the construction of a three dimensional model of TLP-ste that is sufficiently reliable to predict the effects of site directed mutations (Vriend, G. and Eijsink, V. G. H. (1993) J. Comput. Aided. Mol. Des. 7, 367-396.). The 55-69 (referring to FIG. 7, FIG. 7B and SEQUENCE ID No. 2) region was expected to be highly similar in TLP-ste and thermolysin. Comparison of the two known TLP structures, thermolysin and the TLP from B. cereus (Stark, W., Pauptit, R. A., Wilson, K. S. and Jansonius, J. N. (1992) Eur. J. Biochem., 207, 781-791) supported this: TLP-cer has lower homology to thermolysin (73% sequence identity) but, nevertheless has a strikingly similar fold in the 55-69 (referring to FIG. 7, FIG. 7B and SEQUENCE ID No. 2) region (the RMS positional difference is in the order of a few tenths of an Angstrom, that is, in the order of the crystallographic error). Indeed, the TLP-ste model has been used successfully for the rational design of stabilising mutations (Mansfeld, J., Vriend, G., Dijkstra, B. W., Venema, G., Ulbrich-Hofmann,R. and Eijsink, V. G. H. (1995) in: Perspectives on Protein Engineering. (Geisow, M. J. and Epton, R. eds), pp. 205-206, Mayflower, Worldwide Ltd, Birmingham , UK). Structure analyses, three dimensional modelling, prediction of the effects of point mutants, and data base searches were performed with the WHAT IF program (Vriend, G. (1990) J.Mol.Graphics, 8, 52-56). Referring to FIG. 7, described in detail, infra, the only insertion/deletion in the alignment of thermolysin (316 residues) and TLP-ste (319 residues) is a three residue insertion between residues 25 and 30 in TLP-ste. In FIG. 7, this insertion was omitted in the numbering of TLP-ste sequence, meaning that TLP-ste residues are numbered according to the corresponding residues in thermolysin. In the instant application, however, two separate sequences (each with conventional sequence numbering)are shown: (i) FIG. 7A (SEQUENCE ID No. 1) for the TLP-ste (319 residues) and (ii) FIG. 7B (SEQUENCE ID No. 2) for the thermolysin (316 residues).
3. Results and Discussion
We set out to search for several additional stabilizing mutations in the 55-69 area (with reference to FIGS. 7 and 7B and SEQUENCE ID No. 2). One mutation, a Ser(copyright) Pro mutation at position 65 in SEQUENCE ID No. 2 (corresponding to position 68 in SEQUENCE ID No. 1), has been described previously, and did not add anything new to previous strategies. The second and most important mutation concerns a very different strategy, away from modifying the weak region. It concerns the introduction of a disulfide bridge cross-linking residue 60 (in SEQUENCE ID No. 2) and residue 63 (in SEQUENCE ID No. 1) in the critical region with residue 8 in the underlying xcex2-hairpin (FIG. 1). The resulting 8-fold mutated TLP-ste variant is the most stable enzyme ever obtained by protein engineering, with a half life at 100xc2x0 C. of almost 3 hours (Table 1 and FIG. 2).
The half-life of the 8-fold mutant at 100xc2x0 C. was more than 1000 times that of the wild-type and the temperature for optimum activity was raised by 21xc2x0 C. The specific activities at 37xc2x0 C. were identical for the wild-type TLP-ste and the 8-fold mutant (Table 1). In contrast to the wild-type, the 8-fold mutant was active and stable in the presence of high concentrations of denaturing agents (Table 2). The cleavage specificity at both moderate and high temperatures was largely unaffected by the stabilizing mutations (Table 1 and FIG. 3). In summary, the enzymatic properties of the constructed variant resemble those of the wild-type, but its stability resembles that of extremozymes or thermozymes produced by organisms that are capable of surviving in extreme environments such as Archaea and Eubacterial extremophiles.
As an example of the enzymatic activity of the 8-fold mutant we tested the hydrolysis of protease resistant xcex1-amylase from Bacillus licheniformis by the 8-fold mutant. B. licheniformis xcex1-amylase (1 mg/ml) in 50 mM MOPS, pH 7.0, 5 mM CaCl2, 0.01% Triton X-100 was incubated with purified TLP-ste (1 xcexcg/ml), the 8-fold mutant or without protease for 60 minutes at the temperature indicated. The reaction volume was 500 xcexcL. After incubation the samples were cooled on ice, which resulted in aggregation of the substrate in the samples that had been incubated at 100xc2x0 C. Precipitates (only observed in the 100xc2x0 C. samples) were collected by centrifugation and redissolved in 500 xcexcL 6 M Urea. Both supernatants and redissolved precipitates were subjected to standard SDS-PAGE, including pre-treatment with sample loading buffer (5 minutes at 100xc2x0 C.). The samples were identical in size (20 xcexcL supernatant and 20 xcexcL dissolved precipitate). Gels were stained with Coomassie-brilliant blue. No significant degradation of xcex1-amylase occurred at temperatures of 80xc2x0 C. and lower, irrespective of the enzyme used. In case the samples were incubated at 100xc2x0 C. without added protease or with TLP-ste the aggregate formed after cooling contained mature xcex1-amylase, indicating that no hydrolysis had occurred. The B. licheniformis xcex1-amylase that was incubated with the 8-fold mutant at 100xc2x0 C. was completely hydrolysed and no aggregate was formed.
Studies on proteins from extremophiles have revealed that adaptation to extreme environments can normally be attributed to intrinsic properties of these proteins, although in some cases contributions of particular intracellular components, e.g. heat shock proteins and so-called xe2x80x9cthermoprotectantsxe2x80x9d, have been demonstrated. The number of extremozymes for which the structure and sequence could be compared with mesophilic counterparts is limited. On the basis of comparative studies of the primary structures of proteins from mesophilic and thermophilic organisms general rules for stability have been proposed. It has been observed that extremozymes are often less active at lower temperatures than their mesophilic counterparts. The present results, however, lead to the unexpected but important conclusion that it is possible to engineer extremozymes that retain their full activity at lower temperatures.
The present study shows that boiling-resistant proteins can be obtained by rational design. The key to success is insight in the often local and unpredictable unfolding processes that determine the rate of irreversible thermal inactivation. Rationally designed extremozymes can be valuable biocatalysts, as exemplified by the ability of the 8-fold TLP-ste mutant to hydrolyse stable, protease-resistant substrates. With respect to applications, it is most important to note that the 8-fold mutant displays adaptation to extreme conditions without forfeiture of enzymatic performance.
With regard to calcium binding (referring to FIGS. 7A and 7B and SEQUENCE ID No. 2), it was found that from a structural point of view Asp57 seemed more important for calcium binding than Asp59 because both Ods of Asp57 interact with the calcium versus only one Od of Asp59 (FIG. 4). Asp57 was replaced by Ser because in the less thermostable TLPs residue 57 is a serine. From a visual inspection of the three dimensional environment of residue 57 it was concluded that the D57S mutation would not have additional negative effects such as disturbance of the local hydrogen bonding network or the introduction of clashes. To compensate the expected destabilising effect of this mutation, the combined T63F-A69P mutation was chosen. The stabilising mutations had been identified in previous site-directed mutagenesis studies of differences between naturally occurring TLPs (Van den Burg, B., Enequist, H. G., Van der Haar, M. E., Eijsink, V. G. H., Stulp, B. K. and Venema, G. (1991) J. Bacteriol. 173, 4107-4115) and in studies concerning the design of stabilising Xxx- greater than Pro mutations in TLP-ste (Hardy, F., Vriend, G., Veltman, O. R., van der Vinne, B., Venema, G. and Eijsink, V. G. H. (1993) FEBS Lett. 317, 89-92). The mutations are located in the direct environment of Ca3 and the double mutation had previously been shown to drastically stabilise TLP-ste. Characteristics of the various mutants, including the dependence of stability on calcium concentration are presented in Tables 5 and 6 and in FIGS. 5 and 6. As shown in Table 5, the wild-type and mutant enzymes were similar with respect to their activity towards FaGLa.
Referring to FIGS. 7 and 7B and SEQUENCE ID No. 2, the D57S mutation reduced the T50 of TLP-ste at 12.5 mM calcium from 77.9xc2x0 C. to 69.4xc2x0 C. (Table 4). In the stable T63F-A69P mutant the effect of the D57S mutation was even more noticeable, and reduced Tso from 90.2xc2x0 C. to 77.2xc2x0 C. Thus, the integrity of the Ca3 site is clearly important for TLP-ste""s thermal stability.
The stability of TLP-ste and the T63F-A69P mutant (referring to FIGS. 7A and 7B and SEQUENCE ID No. 2) (which both have the Ca3 site intact) depended strongly on the calcium concentration (FIG. 5, Tables 4, 5). Introduction of the D57S mutation reduced this calcium dependence. Consequently, the destabilising effect of the D57S mutant became smaller with decreasing calcium concentration; at the lowest calcium concentration tested, the wild-type enzyme was even slightly stabilised by the D57S mutation). The stability versus calcium concentration curves of TLP-ste and T63F-A69P (FIG. 5) can be superimposed remarkably well. The same is true for the D57S and the D57S-T63F-A69P, strongly suggesting that the observed effects on the calcium stability are indeed caused by the disturbance of the Ca3 site by the D57S mutation.
The D57S-T63F-A69P mutant (referring to FIGS. 7A and 7B and SEQUENCE ID No. 2) represents a TLP-ste variant whose stability is largely independent of the calcium concentration and which, at lower calcium concentrations, is considerably more stable than the wild-type enzyme (Table 5). Combining known stabilising mutations in the Ca3 region has resulted in extremely stable TLP-ste variants. Therefore, it is likely that mutants can be designed that are even less dependent on calcium than the ones described here and that are more stable. Also, and on the other hand, the knowledge now obtained about the calcium-binding site can be used to design variants that are still calcium-dependent while at the same time having obtained a much higher resistance to elevated temperatures or that are even resistant to boiling. Such enzymes can be used in reactions that require prolonged boiling but that can be stopped by changing the calcium concentration in the reaction mixture, by for example adding calcium or chelating agents that capture calcium, depending on the needs of the protease used. Engineering calcium-independence does not necessarily need to be based on detonating the Ca3 site. Instead, it could be based on adding mutations that stabilise the local structure, regardless of the presence of a calcium ion. For example, preliminary analyses of a mutant in which the (intact) calcium binding site is covalently cross linked with the N-terminal xcex2-hairpin showed that the stability of this mutant is also less calcium-dependent.
Comparison of hydrolytic capacities of an 8-fold mutated TLP-ste variant (Boilysin) and commercial enzymes.
In industrial applications enzymes are used for the hydrolysis of protein preparations derived from different sources. Here we show the results of a comparative study in which the rate and quality of hydrolysis of casein and yeast protein preparations by the 8-fold mutated TLP-ste variant was compared with that obtained by the hydrolysis using 3 different enzyme preparations that are used for these purposes in industry. The hydrolysis conditions were chosen such that they were optimal for each of the enzyme preparation under study. The hydrolysis products were separated in an acid-soluble and a not acid-soluble fraction. The amount of acid-soluble protein material formed upon hydrolysis was determined by means of measuring the absorbance at 275 nm, whereas the composition of the acid-insoluble hydrolysis products was studied using SDS-PAGE.
Experimental
Substrate (10% w/v of casein or yeast protein preparation) was incubated in 50 mM Tris-MES, 5 mM CaCl2, pH 6.5 at 50xc2x0 C. (Enzyme I, Favourzyme(trademark) 1000L); 50 mM Tris-MES, 5 mM CaCl2, pH 6.0 at 60xc2x0 C. (Enzyme II, BioProtease N100L); 50 mM Tris-MES, 5 mM CaCl2, pH 8.0 at 60xc2x0 C. (Enzyme III, Alcalase 2.4L); or in 50 mM Tris-MES, 5 mM CaCl2, pH 7.0 at 90xc2x0 C. (Boilysin). Concentration of the enzymes used was 0.05% (v/v). Samples were withdrawn at different time points and transferred to 0xc2x0 C. The solubilized protein fraction was separated from the non-soluble fraction by centrifugation. Acid-precipitable proteins were separated from the acid-soluble fraction by TCA precipitation. The amount of acid-soluble protein released by hydrolysis was determined by measuring the absorbance of the samples at 275 nm. The acid precipitated proteins were redisolved in 6 M urea and subsequently analysed by SDS-PAGE (15% running gel).
Results
FIG. 8 shows the acid-soluble protein (panel A) released upon hydrolysis of casein with the different enzyme preparations in time. In FIG. 1, panel B the size-distribution of the not acid-soluble proteins obtained during hydrolysis of casein is depicted.
FIG. 9 shows the acid-soluble protein (panel A) released upon hydrolysis of yeast proteins with the different enzyme preparations in time. In FIG. 2, panel B the size-distribution of the not acid-soluble proteins obtained during hydrolysis of this protein mixture by the four enzymes is shown.
Conclusion FIGS. 8.A and 9.A suggest that hydrolysis of casein and yeast proteins is more rapidly and that more acid-soluble protein products are produced by the 8-fold mutated TLP-ste variant, as compared to the other enzyme preparations. FIGS. 8.B and 9.B show that a much broader range of hydrolysis products is generated by hydrolysis with the 8-fold mutated TLP-ste variant than with the other enzymes tested.